J. Pesticide Sci. 2, 41-49 (1977)

Effect of the Inclusion Compounds of Pyrethroids and Methyl Parathion on Certain Cotton Insects

Izuru YAMAMOTO, Kanju oxsAWA and Frederick W. PLAPP, Jr.* Department of Agricultural Chemistry, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156, Japan *Department of Entomology, Texas A & M University, College Station, Texas 77843, U.S.A. (Received July 7, 1976)

Methyl parathion is included in beta-cyclodextrin in 1:2 or 1:1 molar ratio. These and the inclusion compounds of four pyrethroids including bioallethrin, tetramethrin, resmethrin, and pyrethrins were examined for their insecticidal activity to certain cotton insects under laboratory conditions. Included methyl parathion (1:2 ratio) was as toxic as free one to Anthonomus grandis, while the per- sistency was remarkably improved, almost all remaining after 10 days on cotton leaves under green house conditions. Inclusion did not much reduce the toxicity of methyl parathion to a parasitoid, Campoletis sonorensis, but the possible reduction in use by inclusion may reduce its side effect to natural enemy of cotton pest insects. Pyrethroids were far less toxic to the parasitoid than methyl parathion and the inclusion further improve the safety characteristic. However, higher doses seemed required for included as compared to free pyrethroids to kill Anthonomus grandis. To Heliothis virescens the included methyl parathion was toxic, but pyrethroids were toxic only by contact, and the insect which fed the included pyrethroids seemed not to be affected.

sistency of pyrethroids is improved. 2) For INTRODUCTION example, under ultraviolet irradiation, the An inclusion compound as referred to here half-life of allethrin was extended by inclusion is a molecular compound between beta-cyclo- from 150 hr to over 500 hr. In talc dust or dextrin and a pyrethroid or methyl parathion. wettable powder formulations under sunlight, Cyclodextrin; which is also called Shardinger allethrin decomposed within 10 to 30 days. dextrin or cycloamylose, is an oligosaccharide In contrast, only slight decomposition of in- obtained from the breakdown of starch by the cluded allethrin occurred within a month. action of Bacillus marcerans or its amylase.1) Pine leaves treated with a 0.1% solution of In beta-cyclodextrin (abreviated as CD) 7 natural pyrethrins or resmethrin gave 100% glucose units are combined by alpha-1,4 link- mortality when fed to pine caterpillars, but ages to make a ring, or donut-like structure. lost activity when tested a week after treat- There is a cavity inside the ring, 7 to 8 Ang- ment. Their inclusion compounds gave 90% stroms in diameter, in which many organic mortality after 2 weeks. The volatility of molecules, as far as size and hydrophobicity dichlorvos was reduced by inclusion.3) A are appropriate, can be included. The included deposit which was lost within 24 hr when kept molecule is often referred to as the guest, and under 2 mmHg at 25C, remained nearly intact CD as the host. when included. When included, the physical, chemical, and Contact insecticidal activity is reduced upon biological properties of the guest compound are inclusion.2) Included allethrin did not show sometimes changed quite remarkably. Per- any contact toxicity to the house fly when 42 日本 農 薬 学 会 誌 第2巻 第1号 昭 和52年2月

assayed as a residue on filter paper. However, methyl parathion to CD was calculated as it was insecticidal when fed to the insect as a 1:2.05. Recovery of methyl parathion in the mixture with sugar. inclusion compound was 68%. Increase of the Pyrethrins and some synthetic pyrethroids charged molar ratio from the above 1:2 (methyl are very toxic to certain forest and agricultural parathion: CD) to 1:3 increased the recovery pest insects, 4,5) but their instability under field to 85%, but the molar ratio in the inclusion conditions limits their use. Based on the finding compound remained 1:2. This product is that including these insecticides seems promis- referred to as methyl paration/CD (1:2). When ing for increasing their longevity in the field, the charged ratio was 1:1, the recovery was we examined the toxicity of included pyreth- 64.3% and molar ratio in the inclusion com- roids to certain cotton insect pests. pound was approximately 1:1. The prepara- A major problem associated with insecti- tion is referred to as methyl parathion/CD cidal control of cotton insect pests such as the (1:1). Table 1 gives samples and the content boll weevil is that the insecticides used also of the active ingredients. kill the natural enemies of Heliothis spp. and spider mites, thus creating secondary pest Table 1 List of pyrethroid and methyl para- problems. Inclusion of the insecticides would thion samples. hopefully alleviate the problem of toxicity to parasites and predators while retaining toxicity to phytophagous pest species. Therefore, we also prepared the inclusion compound of methyl parathion and included it as a reference stand- ard in comparison with the pyrethroids. In addition to tests with pest insects, we ex- amined the toxicity of all insecticides to a beneficial insect, a hymenopterous parasite of Heliot his. MATERIALS AND METHODS The inclusion compounds of pyrethroids were prepared by the method of Yamamoto et al.2) Samples of the inclusion compounds of * Pyrethroid/CD: the inclusion compound of pyrethroids used in the present experiment were prepared and supplied by Teijin Limited, pyrethroid, MP/CD: the inclusion com- pound of methyl parathion, MP: an emul- Tokyo, Japan. Two different types of the sifiable concentrate of methyl parathion. inclusion compound of methyl parathion were ** A micro-encapsulated formulation of meth- prepared. yl parathion. Nylon is the encapsulatory agent. 1. Preparation of the Inclusion Compounds of *** Assumed . Methyl Parathion Five g of technical methyl parathion dis- 2. Analysis of Methyl Parathion Content in solved in 10 ml of acetone were dropped into a the Inclusion Compound stirred suspension of 43 g of CD (about 2 About 150 mg of an inclusion compound were equivalent moles of host to the guest) in one dissolved in one ml N, N-dimethylformamide liter of distilled water. Efficient stirring was at room temperature or 5 ml aqueous methanol needed. After 2 days of stirring, the precipi- (1:1) by warming. Each solution was diluted tate was filtered using a Buchner funnel with with 20 ml water and extracted 3 times with suction, washed with 30 ml of water and air- diethyl ether (15, 10, and 5 ml). The extracts dried. Yield was 33.5 g. By analysis as were dried over anhydrous sodium sulfate and described below, the content of methyl para- concentrated to 25 ml. An aliquot, after dilu- thion was 10.16% and the molar ratio of tion by 1,000 to 10,000 times with petroleum Journal of Pesticide Science 2 (1), February 1977 43

Fig. 1 Residue analysis of methyl parathion on or in cotton leaves treated with the insecti- cide in the form of emulsifiable concentrate, microcapsule, or the inclusion compound in beta-cyclodextrin. ether, was analyzed by a gas chromatograph, in Fig. 1. Microtek Model 110 with a 63Ni detector. 4. Insects 3. Residue Analysis of Methyl Parathion on Heliothis virescens and Campoletis sonorensis or in Cotton Leaves Treated with Various were laboratory reared in the Department of Forms of Methyl Parathion Entomology, Texas A & M University. Boll A conventional procedure was modified to weevils, Anthonomus grandis, were supplied by accomplish the analysis of included methyl the USDA Boll Weevil Research Laboratory parathion as well as micro-encapsulated and Starkville, Mississippi. free methyl parathion. The procedure is shown

Fig. 2 Classification of different application methods of insecticides. 44 日本農薬学会誌 第2巻 第1号 昭和52年2月

5. Application of Insecticides and Bioassay Exp. 5. Leaves of field cotton plants were Methods painted with 0.25 ml of an aqueous sus- Several different procedures were used to pension containing 2.5 mg of toxicant. find satisfactory methods for evaluation of A treated leaf was placed in a 9 cm petri insecticidal activity. A classification of the dish and 5 boll weevils were released. different methods is given in Fig. 2, and details Four replications were made. Only one of the methods are as follows: day samples were available because of Exp. 1. One potted cotton plant was placed heavy rains. at the bottom of a cylinder and sprayed Exp. 6. Leaves of potted cotton plants were from the top with one ml suspension of painted with 0.5 ml of an aqueous suspen- inclusion compound containing one mg sion of inclusion compounds containing pyrethroid or 5 mg methyl parathion. 2.5 mg of toxicant. Non-included com- Four well-sprayed leaves were picked and pounds were dissolved in a small amount each was placed on a moistened filter pa- of acetone and Tween 20, diluted with per in a jelly dish of 9 cm diameter and 5 water to 0.5 ml and painted on leaves. cm depth with 5 boll weevils. The dish Treated plants were placed outside in the was capped by a metallic cap with many daytime and in a greenhouse at night or air holes. Four replications were made. when raining. Bioassays were made at 0 Exp. 2. Leaf disks, 9 cm in diameter, were and 18 days after treatment. A treated cut from field-grown cotton. Each disk leaf was placed in a 9 cm petri dish with was painted with 0.5 ml of an aqueous 5 boll weevils for 0 day samples and 4 suspension of a pyrethroid inclusion com- weevils for 18 day samples. Three repli- pound containing 0.25 mg toxicant. The cations were intended, but certain 18 day dish was placed on a moistened filter paper leaves were not available because of absci- in a 9 cm petri dish and 5 boll weevils were sion of the leaves. released. Three replications were made. Exp. 7. A leaf disk of 9 cm diameter cut from Exp. 3. A 20 ml aqueous suspension of in- field cotton was sprayed with 5 mg toxi- clusion compound or a 20 ml mixture in cant for pyrethroid samples and one mg acetone-water (1:5) of a free pyrethroid toxicant for methyl parathion smaples. containing 2 mg of toxicant was sprayed Free pyrethroids were dissolved in one ml on one field cotton plant encircled by a of acetone-water (1:5) mixture and other box (to prevent drift). From the plant 12 samples were suspended in one ml of leaves were picked and disks of 3.7 cm water. Each treated leaf disk was placed diameter was made from each leaf. Four on a moistened filter paper in a 9 cm disks were placed on a moistened filter petri dish with 5 boll weevils. Three paper in a 9 cm petri dish and 5 boll replications were made. weevils were released. Three replications Exp. 8. Application was the same as that in were made. Sampling was done 2 and 5 Exp. 1. Three Heliothis virescens larvae days after treatment. were released in one jelly dish as in Exp. Exp. 4. Leaves of field cotton plants were 1. Four replications were made. After sprayed with one ml solution or suspension spraying the plants were held outside of sample through a cylinder of 9 cm dia- under a roof for protection from rain, but meter. Each spray contained one mg still exposed to direct sunlight for several toxicant. The inclusion samples were sus- hours a day. Sampling was made at 0, 7, pended in water, while free pyrethroids and 10 days. were prepared in acetone-water (1:5). The Exp. 9. In Exp. 9A the whole surface of a sprayed portion of the leaf was excised and leaf was painted with 0.5 ml of an aqueous placed in a 9 cm petri dish with 5 boll suspension of an inclusion compound con- weevils. Three replications were made. taining 0.25 mg of toxicant. Each treated Only one day samples were available be- leaf was placed in a 9 cm petri dish and 2 cause of heavy rains. H. virescens larvae were released. Three Journal of Pesticide Science 2 (1), February 1977 45

replications were made. Many leaves des- In all experiments, toxicity measurements sicated and cannibalism occurred in several were based on 3 day data if not otherwise dishes. In Exp. 9B a leaf disk of 9 cm specified. diameter was painted and the leaf disk was placed on a moistened filter paper in RESULTS AND DISCUSSION a petri dish to avoid dessication of the 1. Contact Insecticidal Activity to A. grandis disk. and H. virescens (Table 2) Exp. 10. A leaf disk of 9 cm diameter cut Remmethrin and its inclusion compound from field cotton was sprayed with one were used as a representative of pyrethroids. mg of toxicant in one ml of an aqueous Free resmethrin gave 100% mortality to A. suspension for the inclusion compound of grandis at a 1 mg dose, while the inclusion pyrethroids or acetone-water (1:5) mix- compound did not show any activity at a 2 mg ture for the free pyrethroids. The leaf disk (a. i.) dose. Free resmethrin was toxic to H. was placed on a moistened filter paper in a virescens at 0.1 mg and higher doses. The 9 cm petri dish with 3 Heliothis larvae, included preparation gave no mortality at a 1 which were sectioned by a paper divider mg dose and a slight mortality at a 2 mg dose. to minimize cannibalism. One replication There was no mortality of these insects on un- was made because of the shortage of in- treated filter paper or filter paper treated with sects. 14.7 mg of CD (2 mg of free resmethrin which Exp. 11. Spraying and sampling were the are included in 14.7 mg of CD compounds). same as in Exp. 3, except 10 mg toxicant The result indicates that upon inclusion, con- in 15 ml were used. Four Heliothis larvae tact insecticidal activity is greatly reduced. and 4 leaf disks were placed in a petri dish and sectioned by a paper divider to avoid 2. Contact Insecticidal Activity to a Hymeno- cannibalism. Three replications were pterous Parasitoid, Campoletis sonorensis made. (Table 3) Exp. 12. To evaluate intrinsic contact In- Under the same experimental conditions as secticidal activity a 9 cm filter paper was described above, free resmethrin, pyrethrins, treated with one ml of acetone solution of and bioallethrin gave 100% mortality at a 1 free insecticide or one ml of aqueous sus- mg dose, while their inclusion compounds did pension of the inclusion compound and not kill the insect at the same dose. Tetra- air-dried overnight for the latter. Each methrin was less toxic. Contrary to pyreth- treated filter paper was placed in a petri roids, methyl parathion was quite toxic to the dish and 5 boll weevils, 4 Heliothis larvae, insect. Although inclusion reduced the toxicity or 2 Campoletis adults were released. The to some extent, it was still high. Free methyl Heliot his larvae were sectioned by a parathion killed the insect at a 0.5 ag dose and divider, and each was given a small amount the inclusion compound gave partial mortality of the conventional diet. from the 0.5 to 5 ug level.

Table 2 Toxicity of free and included resmethrin to adult A, grandis and larval H. virescens by a filter paper contact method.

* Killed/released insects . 46 日本 農 薬 学 会 誌 第2巻 第1号 昭 和52年2月

Table 3 Toxicity of pyrethroid and methyl parathion samples to adult C. sonorensis by a filter paper contact method.

* Killed/released insects .

3. Application of Insecticides to Cotton Plants complete kill when observed 4 days after release for Bioassay of the insects. However, there was a distinct Painting (Exps. 2, 5, 6, 9) gave a far better difference of speed of action between free and coverage than spraying and the dose applied included pyrethroids, the latter being slower. could be better controlled. However, painting A similar tendency can be seen in Exp. 6 with sometimes caused injury to the leaves, was leaves sampled 0 days after treatment. The very laborious, and was not always quantita- results with 18 day leaves of Exp. 6 are some- tive because the applied aqueous suspension what dubious, because known unstable pyreth- sometimes dripped from the leaves before roids such as tetramethrin and resmethrin gave drying. When cotton plants were sprayed all high mortality. Abscision of many 18 day over, there were differences in toxicant amount cotton leaves resulted in the less available among treated leaves. In Exps. 1 and 8, four replications in this experiment which may well-covered leaves were used for assay, while increase the variation of the results. At lower in Exps. 3 and 11, small leaf disks were made doses toxicity was reduced for both free and from many treated leaves for randomization. included pyrethroids. There are some contra- Application of insecticide on a defined area of dictions among the results. The included cotton leaf by a sprayer through a cylinder (in resmethrin and pyrethrins seemed less effective this case, 9 cm diameter) was feasible both for than others in Exp. 6, but they were more leaves on plants and after excision, and gave active than others in Exps. 2 and 1. Perhaps uniform coverage of insecticide on the treated deviation of the results at lower doses was too leaf (Exps. 4, 7, 10). Results obtained by such large to give consistent results. Permethrin different methods will not be compared di- was the only pyrethroid which gave 100% rectly. For convenience, however, estimated mortality at a 0.5 kg/hectare level, and con- dose in terms of kg/hectare is given in Table sistently gave good results at higher doses. 4 based on the assumption that 88,000 cotton Slower action or perhaps lower activity of plants were standing per hectare. the inclusion compounds of pyrethroids as compared to free pyrethroids may be overcome 4. Effects on A. grandis if the former have longer persistency. How- Considering the adequacy of application ever, persistency tests were mostly failures procedure of insecticides and other factors, we because of rainfall. Exp. 3 lists the only actual may rely most on the result in Exp. 7. At the data on plants treated in the field and not high dose of approximately 2 kg/hectare, sampled immediately. Because of the low dose, pyrethroids were quite toxic, whether free or however, the results were not useful in terms included, to the boll weevil. Most gave almost of persistency. Journal of Pesticide Science 2 (1), February 1977 47 48 日本 農 薬 学 会誌 第2巻 第1号 昭 和52年2月

Table 5 Persistency of methyl parathion in important. In our bioassay conditions, there various forms on cotton leaves un- was almost no feeding on leaves treated with der sunlight as measured by gas active free pyrethroids, but there was heavy chromatography. feeding on leaves treated with less active free pyrethroids and included pyrethroids. There- fore, detoxication in the gastrointestinal system of the insect must be the reason for the ineffec- tiveness of the included pyrethroids as stomach poisons. There was a surprising result with included resmethrin in Exp. 8. It was as effective as methyl parathion and more persistent, even at the 0.1 kg/hectare level. Free resmethrin and permethrin seemed to be good contact Experimental conditions: A 1 ml aqueous insecticides to the insect. Included methyl suspension of the sample containing 1 mg methyl parathion was placed on a leaf of parathion was highly effective except in Exp. potted cotton and kept under sunlight, but 9B. Whether it is active as a stomach poison protected from rain. or a contact poison is not known. In conclusion, although there are some con- Methyl parathion was effective in any form. tradictory or incompatible data present, we We anticipated a lower activity for the in- partly determined the basic toxicity of the cluded methyl parathion, but actually there inclusion compounds of pyrethroids and methyl was no difference among emulsifiable concen- parathion in relation to cotton and certain trate, microcapsule, and inclusion compound. cotton insects. The results provide information All gave 100% mortality, at a 0.5 kg/hectare to be utilized for further investigations on level. The result of residue analysis after one cotton insect control. hour and 10 days is shown in Table 5. Free Increased persistency of methyl parathion methyl parathion was quickly lost from the and reduced contact activity of pyrethroids surface of cotton leaves. Only 54% remained by inclusion were shown. All included insecti- after one hour and none was left by 10 days. cides were toxic to A. grandis, although higher On the other hand, persistency of methyl doses seem to be required for included as parathion included in CD in 1:1 or 1:2 ratios compared to free pyrethroids. To H. virescens was excellent. The 1:1 inclusion compound pyrethroids are toxic only by contact and the seems to be a inbetween of free and completely included pyrethroids are generally less effec- included methyl parathion, for it gave residue tive, probably because of detoxication in the data between free and 1:2 inclusion methyl gastrointestinal system. Included methyl parathion. Microencapsulated methyl para- parathion is as toxic as free methyl parathion thion gave a higher persistency than free to A. grandis, while persistency is remarkably methyl parathion, but it was not comparable improved, thus giving a possibility to reduce to the included preparations. In view of the the amount of the insecticide needed for boll results, persistency of included methyl para- weevil control. thion is not due to its slow release, but probably Pyrethroids are far less toxic to a parasitoid, due to reduction of volatility and photodecom- C. sonorensis, than methyl parathion. Inclusion position. further improves the safety characteristic. The inclusion of methyl parathion does not 5. Effect on H. virescens much reduce its toxicity to the parasitoid, but From Exps. 10 and 11, it seemed that free the possible reduction in use by inclusion may pyrethroids were generally more toxic than reduce its side effect to a certain extent. the included ones. In the field, where H. virescens do not usually eat cotton leaves, contact action of insecticides is said to be more Journal of Pesticide Science 2 (1), February 1977 49

パ ラ チオ ン(l:2比)はAnthonomus grandis(ワ タ ミ ACKNOWLEDGEMENTS ゾ ウ ム シ)に 対 し遊 離 メ チ ルパ ラ チ オ ン と同等 の効 果 を Approved for publication by the Director, Texas Agricultural Experiment Station. This 示 す 一 方, そ の 残 効 性 は き わ め て長 くな り, 温 室 内 で 棉 publication was supported in part by the Na- 植 物 の 葉 上10日 後 で も ほ とん ど 分 解 しな か った. しか tional Science Foudation and the Environmental し寄 生 蜂 の 一 種Campoletis sonorensisへ の毒 性 は包 Protection Agency, United States of America, 接 化 に よ って あ ま り減 ら なか っ た. ピ レス ロイ ドは いず through a grant (NSF GB-34710) to the Univer- sity of California. Additional support was ob- れ もC. sonorensisに 低 毒 性 で あ り, 包 接 化 に よ りさ ら tained from the United States Department of に安 全 性 が 高 め られ るがA. grandisの 殺 虫 に は包 接 物 Agriculture, Regional Research Project S-73, は 遊 離 ピ レス ロイ ドよ り高 濃 度 が 必 要 の よ うで あ る. "Pesticide Residues in Agricultural Commodi- Heliothis virescensに 対 し包 接 メチ ルパ ラ チオ ンは 有 効 ties and Environments." The authors gratefully acknowledge the assistance of Mr. W. H. Vance, で あ った が, 包 接 ピ レ ス ロ イ ドは 十 分 摂 食 され てい て も Texas A & M University, for GLC analyses and 無効 で あ った. Dr. D.L. Bull, USDA, College Station, Texas for advise and assistance with certain bioassays. We REFERENCES especially thank Dr. C. B. Huffaker, University 1) D. French: Advan. Carbohyd. Chem. 12, of California, Berkeley, and Dr. P. L. Adkisson, 189 (1957) Texas A & M University, for help in making 2) I. Yamamoto, T. Unai, Y. Suzuki & Y. necessary arrangements. We also thank Dr. A. Katsuda: J. Pesticide Sci. 1, 41 (1976); Mifune, Teijin Limited and Dr. Y. Katsuda, Presented at the 18th Ann. Meeting of Dainippon Jotyugiku Co., Ltd. for helpful dis- Japanese Soc. Appl. Entomol. Zool., Sapporo, cussions. August, 1974 3) A Mifune: J. Synth. Over.Chem. Japan 32, 要 約 889 (1974)

メ チ ル パ ラ チ オ ン は β-シ ク ロ デ キ ス ト リ ン に 包 接 さ 4) W.H. Lange & N. B. Akesson: "Pyrethrum. The Natural Insecticides," ed. by J. E. れl:2ま た は1:1の 包 接 モ ル 比 を 与 え た. メ チ ル パ ラ Casida, Academic Press, New York and チ オ ン と4種 の ピ レ ス ロ イ ドbioallethrin, tetrame- London, p. 261, 1973 thrin, resmethrin, pyrethrinsの 包 接 化 合 物 の 棉 畠 昆 5) R. P. Miskus & R. L. Lyon: ibid. p. 281,

虫 に 対 す る 室 内 殺 虫 効 果 を 検 討 し た と こ ろ, 包 接 メ チ ル 1973